PROJECT SUMMARY/ABSTRACT Insulin-degrading enzyme (IDE) is an evolutionarily and structurally distinctive zinc-metallopeptidase that is the best characterized mediator of insulin catabolism in vivo. IDE is strongly implicated in the pathogenesis and potential treatment of type 2 diabetes mellitus (T2DM) as well as other highly prevalent diseases. For more than 60 years, pharmacological inhibition of IDE has been viewed as an attractive way to promote endogenous insulin signaling and thereby treat T2DM, and the feasibility of this fundamental idea was recently confirmed in vivo (Maianti et al., Nature 2014). Despite the considerable promise of IDE inhibitors as experimental probes and as potential pharmacophores, only a limited number of inhibitors have emerged. Existing inhibitors do not possess the properties needed to address a large number of outstanding questions about the fundamental biology of IDE, such as the differing roles of intracellular and extracellular pools of IDE. Moreover, current IDE inhibitors lack appropriate medicinal properties, such as oral bioavailability and long-term stability in vivo, needed to be developed into drugs for clinical use. The objective of this proposal is to use fragment-based drug design (FBDD) to develop a diverse set of potent, selective, small-molecule, zinc-targeting IDE inhibitors suitable for use as experimental probes and/or as lead compounds for future drug development. Specifically, in Aim 1, we will screen several custom libraries of metal-binding pharmacophores (MBPs), synthesized in-house, to identify novel chemical moieties targeting the catalytic zinc atom within the active site of IDE. In Aim 2, we will synthesize sublibraries of compounds containing these zinc-targeting moieties; test them for potency, selectivity and other properties; then repeat this process until IDE inhibitors with the desired set of features are obtained. In Aim 3, we will characterize key properties of developed inhibitors, using a battery of in vitro, cell-based, and in vivo assays. An innovative goal of this project is to develop compounds that are, to the greatest extent possible, selective for different substrates of interest, a novel feature of IDE that results from its unusual tertiary structure and atypical mechanism of substrate binding. Accordingly, throughout all phases of the project, we will test all compounds against multiple substrates in parallel. Using this approach, we have so far identified >25 structurally diverse MBPs that inhibit IDE with remarkable potency. Moreover, through the optimization of just a subset of identified MBPs, we have already succeeded in developing drug-like compounds with nanomolar potency. Notably, these novel compounds selectively inhibit the degradation of insulin and also show strong specificity for IDE versus numerous other proteases. The successful completion of this project will result in novel IDE inhibitors suitable for diverse experimental and medicinal applications.